The primary goal of the proposed research is to determine the biological significance of various DNA reaction sites of the antitumor drug, cis-dichlorodiammine platinum. Initial studies will be confined to measuring the template activity of platinum-treated polynucleotides, and DNA having varying G-C mole percent in an assay system employing mammalian DNA polymerase alpha, beta and gamma. The RB-50, the amount of bound platinum required to decrease template activity by 50 percent per molecule of a given molecular weight (measured by a chromastix CMX-4), will be evaluated for each template. Comparison of the RB-50's of the polynucleotides and in DNA with varying G-C mole percent will establish which DNA reactions most significantly lead to template inactivation. The trans isomer, which is a toxic, inactive analogue, will be studied to determine why antitumor activity resides only in the cis configuration. To evaluate the possible contribution of bifunctional DNA reactions to template inactivation, the effects of the monofunctional compound, (diethylenetriamine chloroplatinum) chloride will also be investigated. The effects of platinum on the secondary and tertiary structures of DNA will be determined, and experiments will be performed to test the hypothesis that cis-PT (II) creates interstrand cross-links between opposite and adjacent adenines in DNA. The in vitro experiments are only a prelude to the same measurements in vivo using DNA isolated from treated HeLa cells, or from treated HeLa cell nuclei. Particular emphasis will be placed on analyzing the yield of various DNA binding sites from in vivo treatment. Template activity of DNA from treated HeLa cells will be assayed using the same DNA polymerases, or treated DNA in isolated nuclei would be assayed by endogenous enzymes for template activity. Then the template activity associated with a given level of DNA binding would be correlated with macromolecular synthesis inhibition and cell kill. Finally, the possible role of ineffective template inactivation in cultured cells which are resistant to cis-PT(II) but not resistant to cyclohexyldiamine malinato platinum (CHAMP), an active analog of cis-PT(II) will be compared with results obtained in cells resistant to CHAMP, but not resistant to cis-PT(II).